Exploring histopathological and serum biomarkers in lung adenocarcinoma: Clinical applications and translational opportunities (Review).

Lung cancer is the second most common neoplasm in the world, but the first in males, and the leading cause of cancer-associated death. In 2020, there were >2.2 million new cases globally, causing >1.8 million deaths ( 1 ). Despite the remarkable advances made in the diagnosis, immunotherapy and monitoring of the disease, the average 5-year survival rate is ~23-27%. Lung cancer is characterized as having a high lethality and comorbidity, and the majority of patients are diagnosed in advanced stages where curative surgical options are limited ( 2 ). It should also be noted that mortality data are similar in developed and emerging countries. Even for patients who underwent curative surgery for the clinical management of localized tumors, the 5-year survival rate is only 65%, demonstrating the aggressiveness of these tumors despite those patients having been diagnosed in early stages. In addition, >70% of patients diagnosed with lung cancer have locoregional or metastatic lymphatic spread, decreasing the probability of survival at 5 years to ~33 and ~7%, respectively ( 3 ). In developed countries, the incidence of lung cancer has decreased in recent years thanks to measures to prevent tobacco use and control occupational exposure to asbestos, since the former represents the main risk factor in the general population. These primary prevention measures have caused a 45% decrease in lung cancer mortality in males in the 1990-2015 period and a 19% decrease in females in the 2002-2015 period ( 4 ). Other risk factors for lung cancer have been described and include a multitude of agents such as radon, arsenic, benzopyrenes, asbestos, infections such as tuberculosis, and environmental pollution, although their contribution to global cases is only minor, as tobacco smoking is responsible for up to 90% of lung cancer cases ( 5 ). This is due to the fact that combustion of tobacco causes the release of polycyclic hydrocarbons, nitrosamines, nitrates and 60 other carcinogens, which induce alterations in DNA repair mechanisms, cell cycle control and dysplasia processes that lead to histological degeneration, followed by predominating proliferation and invasion of aberrant malignant cells ( 6 ). Smoking cessation is associated with a clear decrease in the relative risk of developing lung cancer after 10-15 years ( 7 ). It should be noted that ~1.1 billion individuals smoke in the world and of these, 10-20% of smokers may develop lung cancer ( 8 ). On the other hand, lung cancer is more frequent in males and the maximum incidence by age is between 80 and 90 years ( 9 ). The histological varieties of lung cancer have been traditionally classified by prognostic, pathological and therapeutic factors. They have been differentiated into small cell lung cancer (SCLC) characterized by small cells with a very poor prognosis and mostly associated with paraneoplastic syndromes (Cushing's syndrome, Syndrome of inappropriate antidiuretic hormone secretion) and non-SCLC (NSCLC), which are subdivided into squamous cell carcinoma, large cell carcinoma and lung adenocarcinoma ( 10 ). The subtype most associated with tobacco exposure is squamous cell cancer, but the most frequent subtype is lung adenocarcinoma, which accounts for >60% of non-small cell tumors ( 11 ). Lung adenocarcinoma has unique histological, radiological, epidemiological, molecular and clinical characteristics compared with other tumors. For instance, the incidence of lung adenocarcinoma is similar in males and females, and it is the lung neoplasm with the highest incidence in individuals who have never smoked and <45 years of age ( 12 ). Histologically, lung adenocarcinoma is composed of bronchial glands with a tendency to papillary configuration that degenerate due to deterioration of type II pneumocytes, generating atypical alveolar hyperplasia and later a truly invasive neoplasia. At the pathological level, the 2021 World Health Organization (WHO) classification allows lesions to be differentiated according to their invasive potential, classifying them into minimally invasive mucinous or non-mucinous lesions, and invasive non-mucinous adenocarcinoma, which in turn may be subclassified in acinar, papillary, micropapillary and solid tumors. Other less frequent types include invasive mixed mucinous lesions, colloid adenocarcinoma, fetal or enteric type, each of them with diagnostic, prognostic and clinical peculiarities ( 13 ). Regarding the early diagnosis of this disease, it should be noted that there are screening programs for smokers that have been evaluated by low-dose computed tomography (CT) of the chest as approved by the US Preventive Task Force, but in clinical practice, they are difficult to apply, which means that most patients are diagnosed in advanced stages of the disease ( 14 ). With respect to the clinical management of pulmonary nodules, evaluation with CT, thoracoscopy, mediastinoscopy and positron emission tomography-CT have allowed to improve the diagnosis in the initial stages in these patients, which still represent a small proportion of them and it is associated with a complex management, subjecting the patients to a great level of emotional stress with a follow-up that may last several months ( 15 ). Unlike other tumors, such as pancreatic adenocarcinoma, ovarian, breast and testicular neoplasms, where markers such as CA19-9, CA125, CA15.3 or carcinoembryonic antigen (CEA) may be used, there are currently no serological markers in daily clinical practice that may help diagnose lung cancer ( 16 ). Of note, most patients initially present with constitutional syndrome, hemoptysis or cough. It is also common for numerous patients to present with superior vena cava syndrome, Horner's syndrome, compression of the brachial nerve or pericardial effusion ( 17 ). Although it is true that SCLC is the lung neoplasm that has been most clearly associated with paraneoplastic syndromes, patients with lung adenocarcinoma may present with acanthosis nigricans, dermatomyositis or and Trousseau syndrome ( 18 ). The therapy of these tumors is different according to the time-point of diagnosis. In initial stages (I, II and IIIA), where the tumor is susceptible to surgical treatment, patients may undergo radiotherapy, neoadjuvant chemotherapy and subsequent surgery if they are surgical candidates. In more advanced stages, such as IIIB and IV, where there is mediastinal or subcarinal involvement, contralateral pulmonary invasion or metastatic spread, they are treated with chemoradiotherapy. Furthermore, it is possible to perform a histopathological study in order to administer specific immunotherapy regimes ( 19 , 20 ). In this sense, the use of immunotherapy has been a real advance in recent years as it has improved the prognosis in patients with disseminated disease, but even so, >80% of patients diagnosed with lung adenocarcinoma in the advanced stage do not survive for >5 years ( 21 ). Similarly, the lack of early diagnosis or serological markers is a real challenge in the early diagnosis of this disease. Hence, the purpose of the present review was to discuss the main immunohistochemical and diagnostic markers that not only help in the initial staging but may also be useful in the follow-up of patients in both advanced and early stages. Furthermore, the state-of-the-art of potential serological markers used in these patients was equally revisited, including promising approaches such as circulating tumor cells (CTCs), microRNAs (miRNAs/miRs) and exosomes.

MiRNAs are small non-coding RNA molecules with a length of ~20 nucleotides that regulate the post-transcriptional expression of genes that may be related to cell differentiation, proliferation and apoptosis processes by promoting or suppressing the expression of a gene after transcription. A miRNA molecule regulates the post-transcriptional expression of up to 200 different genes and its study may expand the understanding of the underlying pathophysiology of the metastatic process ( 92 ). In relation to lung cancer, the implications of miRNAs are numerous-they may promote processes such as cellular proliferation, metastatic invasion and therapy resistance through the upregulation or downregulation of either tumor suppressor genes or oncogenes ( 93 ). As previously mentioned, tobacco is the main cause of lung cancer in the general population and it has been indicated how the levels of miR-532-5p, miR-25-3p and miR-133a-3p were significantly higher in patients with lung carcinoma compared to healthy controls, also observing differences between expression levels depending on the smoking status ( 94 ). On the other hand, Nymark et al ( 95 ) indicated how numerous miRNAs are dysregulated according to exposure to asbestos and its relationship with lung cancer. Likewise, there are numerous miRNAs that have been implicated in EGFR mutations, such as miR-7, miR-27a-3p, miR-30 and miR-34, which led to the activation of the RAS/MEK and PI3K/mTOR pathways with consequently uncontrolled cell proliferation ( 96 , 97 ). Similarly, a role of miR-96 has been described in the altered levels of ALK and the activation of the RAS/MEK and PI3K/mTOR metabolic pathways ( 98 ). miR-760 causes alterations in the expression of ROS1, while let-7, miR-193a-3p or miR-148a-3p are related to KRAS mutations, previously demonstrating their importance in tumor progression ( 99 - 102 ). The diagnostic utility of miRNAs has also been studied by different authors. For instance, the presence of miR-205 was specific for squamous cell carcinoma compared to miR-124a, which is more characteristic for lung adenocarcinoma ( 103 , 104 ). The relationship between histology and miRNA expression has made it possible to demonstrate how miR-93, miR-221 and miR-30e are specific for squamous cell carcinoma, while miR-29b, miR-29c, let-7e and miR-125a-5p are more specific for lung adenocarcinoma ( 105 ). One of the uses of miRNAs is based on the possibility of them being used in screening programs that determine the blood levels of multiple miRNAs, which simplifies the diagnostic process as well as its ease of performance and improves its diagnostic performance. Studies such as that by Montani et al ( 106 ) have evaluated the use of a kit with 34 miRNAs in 1,115 individuals with a high risk of lung cancer, obtaining a sensitivity of 75.9%, a specificity of 77.8% and an AUC for the diagnostic yield of 85% ( 106 ). These results are in agreement with those obtained by Sozzi et al ( 107 ), who analyzed 69 patients with lung cancer using a kit of 24 miRNAs, obtaining a sensitivity of 87% and a specificity of 81% ( 107 ). Asakura et al ( 108 ) reported that after analyzing up to 2,588 miRNAs in 208 patients with lung cancer compared to healthy controls, the highest diagnostic yield using miRNAs was obtained with miR-1268b and miR-6075, obtaining a sensitivity and specificity of 99% and an AUC of 0.993 for lung cancer screening ( 108 ). On the other hand, the expression of different miRNAs has been studied to analyze its relationship with the prognosis of patients. Xiao et al ( 109 ), in a meta-analysis of 15 studies that included a total of 1,753 patients with both SCLC and NSCLC, described that upregulation of miR-125b, miR-21, miR-141, miR-200c, miR-197, miR-41, miR-370, miR-376α, miR-192 and miR-662 and the downregulation of miR-26b, miR-381, miR-146α, miR-148α, miR-204, miR-374α, miR-638 or miR-148b were associated with poor median survival, evidencing the complex role of miRNAs in lung cancer. It should be noted that alterations in different miRNAs have been related to mechanisms of chemoresistance and sensitivity to immunotherapy. For instance, overexpression of miR-106b leads to a decrease in the P-glycoprotein responsible for chemoresistance mechanisms to cisplatin, which causes greater sensitivity to cisplatin ( 110 ). In turn, Qiu et al ( 111 ) have demonstrated that downregulation of miR-503 alters the expression of proteins related to chemoresistance processes, such as the antiapoptotic protein Bcl-2, while another study indicated that overexpression of miR-196a leads to decreased efficacy of cisplatin ( 112 ). Given that in recent years, immunotherapy has laid a foundation for the management of patients with lung cancer, the expression of miRNA in this context has been evaluated in numerous studies. For instance, Bisagni et al ( 113 ) examined 32 patients with lung adenocarcinoma receiving second- or third-line treatment with erlotinib, an EGFR tyrosine kinase inhibitor, and miR-133b upregulation was associated with better progression-free survival. However, the main limitation of miRNAs in lung cancer in terms of their usefulness for screening is their limited specificity. For instance, miR-21-5-p, miR-155-5p and miR-210-3p are expressed in different neoplasms, such as breast or colon cancer, among others, which would require patients to undergo multiple diagnostic tests with the probability of adverse effects without a clear diagnostic suspicion. In addition, both upregulation and downregulation of the same miRNA may be observed in different neoplasms, which increases the diagnostic uncertainty. In addition, large clinical trials should be implemented to specifically validate detection kits that are cost-efficient in different neoplasms so that they may be systematically applied in different malignant neoplasms ( 114 ). Examination of miRNAs, which may be performed by liquid biopsy in peripheral blood, has useful implications in the diagnosis, follow-up and treatment of patients with pancreatic adenocarcinoma that may improve diagnosis in early stages and improve the understanding of the mechanisms of immunochemical resistance in these patients.

Lung cancer is one of the most frequent neoplasms and the deadliest type of cancer, which is specifically associated with tobacco consumption. Despite numerous efforts and screening programs that have been performed, most patients are diagnosed in the advanced stages of the disease. Lung adenocarcinoma is a specific subtype of NSCLC with unique histological, radiological, epidemiologica and clinical characteristics. Recent advances in the molecular biology of these tumors have permitted the identification of multiple markers, as summarized in Fig. 1 . The study of these markers has allowed the development of numerous targeted therapies, also aiding to improve the prognosis, diagnosis and prediction of the response to different therapeutic regimes. Likewise, numerous serological markers have been studied, demonstrating promising translational uses. The main markers studied with their most important translational/clinical applications are summarized in Table I . Overall, there is still much to explore in the field of biomarkers in lung adenocarcinoma, particularly regarding the aim to improve early detection of the disease and identifying new molecular routes that may be used for targeted therapies, which is proving to be one of the most important advances in the field of oncology.